Stem cell jackpot for Parkinson's disease

A Memorial Sloan-Kettering Cancer Center team has
honed a protocol for producing large quantities of human dopaminergic neurons
that could be used therapeutically by grafting them into patients with
Parkinson's disease or as a platform for drug screening for the disease.1
But scaling up the protocol remains a challenge, and therapeutic applications
face major regulatory issues due to potential safety concerns.

Parkinson's
disease (PD) is caused by degeneration of dopaminergic neurons throughout the
brain, the effects of which are felt most acutely in the substantia nigra, a
midbrain region involved in movement.

PD
symptoms are treated with l-dopa, a dopamine precursor, but due
to side effects, limited efficacy and the inconvenience of frequent dosing with
this compound or other dopamine agonists, research has turned to finding a way
to restore dopamine levels using neuronal implants.

Since the late 1980s, researchers have tried to replace
dying neurons of the substantia nigra with dopaminergic cell grafts from
aborted fetuses. But despite long-term engraftment and modest clinical
efficacy, the limited availability of source material means that the approach
cannot be scaled up.

Thus,
"there has been an effort to increase the yield of dopaminergic cells in
cell culture prior to transplantation," said Curt Freed, division head and
professor of medicine at the University of Colorado Denver School of Medicine, who conducted the
first fetal cell grafts in PD patients.

Human embryonic stem cells (ESCs), which can be cultured
indefinitely, are potentially a scalable alternative to primary fetal tissue.
However, obtaining high yields of stable dopaminergic cells without also
producing unwanted nondopaminergic cells has been a challenge.

Now,
a team led by Lorenz Studer, professor of developmental biology and director of
the Center for Stem Cell Biology at Sloan-Kettering, has optimized an ESC
culture procedure that yields large quantities of precisely the kind of
dopaminergic cells needed to treat PD.

"The
idea of stem cells for PD is not new, but we've never had a good source of
enriched dopaminergic cells for transplantation," said Studer.

Cells well

Studer got a hint of how to obtain the
dopaminergic cells from his team's earlier efforts to coax ESCs into forming
various neuronal precursors. In one of those prior studies, the team identified
markers for midbrain dopaminergic neuron precursors and developed a procedure
to grow a specialized subset of those cells, called floor plate precursors, in
vitro.2

In
the new study, Studer's team converted the floor plate cells into functioning
midbrain neurons by simultaneously manipulating several signaling pathways that
influence neuronal development.

"The
way to go from stem cells to highly specialized nerve cells is to give a series
of instructions for differentiation," said Studer. The key signal to make
the right kind of neurons turned out to be activation of the wingless-type MMTV integration site (WNT) signaling pathway.

In
cell culture, the ESC-derived neurons behaved like natural dopaminergic
neurons, secreting more dopamine and lower amounts of other neurotransmitters
like serotonin and g-aminobutyric acid (GABA) than neurons generated by previous in
vitro methods. Immunohistochemical analysis showed that Studer's method
also produced more dopaminergic neurons than other types of neurons.

Next,
Studer's team transplanted the in vitro-generated dopaminergic neurons
into mouse, rat and monkey models of PD and found that the neurons successfully
engrafted, survived indefinitely and restored dopaminergic activity in the
midbrain. PD animals receiving Studer's dopaminergic neuron grafts had higher
midbrain dopaminergic neuron density and better performance in gait assays than
animals given dopaminergic neurons made by prior methods.

Studer's
dopaminergic neuron grafts appear to be stable over the long term. Mice
receiving these cells stably expressed dopaminergic cell markers, showed no
signs of contaminating cell overgrowth and showed improved performance in an
assay of amphetamine-induced motion disorder as late as 16 weeks after
transplantation compared with controls receiving neuronal preparations made by
prior methods.

Raising
yield

Academic experts polled by SciBX said
that from a technical standpoint, Studer's new method is an incremental advance
over previous methods, but the resulting increase in efficiency is potentially
a game changer for manufacturing dopaminergic cells.

"The
novelty of the report is the accomplishment of a differentiation protocol that
more reliably generates the correct cell type," said Ole Isacson,
professor of neuroscience and neurology at Harvard Medical School. "This is
definitely an improvement on prior protocols reported by the same group a few
years ago."

Isacson
noted that his own team recently reported that transplantation of mouse
ESC-derived midbrain dopaminergic neurons isolated by fluorescence-activated
cell sorting (FACS) can ameliorate a rat model of PD.3 Studer's
method provides a potential source of large numbers of such neurons without the
need for time-consuming and inefficient FACS protocols.

Although
about 80% of the cells in Studer's in vitro preparation are dopaminergic
neurons, it is not clear what fraction of the starting ESCs are converted into
neurons by Studer's complex procedure.

"The
next challenge is to achieve sufficient yield," said Isacson. "It's
not clear whether they've generated more neurons overall or a higher percentage
of the right neurons."

Studer's method could solve several challenges to ESC
therapies for PD, including concerns about graft purity and the potential for
tumor formation.

Freed
and Isacson said that previous ESC culture methods ran the risk of generating
unwanted cells, in particular serotonergic neurons, which can interfere with
the activity of dopaminergic cells. In contrast, Studer's team showed that
neurons cultured according to the new protocol did not secrete serotonin.
Indeed, at 4.5 months after transplantation, mice receiving grafts grown by
Studer's new method had very few transplanted cells with markers of
nondopaminergic cell identity compared with mice receiving conventional neural
grafts.

Freed
said that for regulators to accept the cells as therapeutic candidates,
long-term preclinical safety studies would be needed to exclude the possibility
of tumor formation. Freed said that in contrast to his original fetal cell
graft studies, which at the time did not fall under FDA regulation, today the
regulatory environment for cell therapies is much more stringent.

"A
renegade stem cell is a potential disaster," said Freed. "FDA might
say that because Studer didn't report much data about tumors, they would like
to see 100 mice for a year who have no tumors with this preparation."

"We
now have right cells," said Studer. "The question is now how to make
these cells in a format that's safe to use" in the clinic. Doing so would
require making the cells under GMP conditions, which would likely require
collaboration with a cell manufacturing company, he added.

Cell side

Cell therapies for PD have not made much
headway since the cessation of fetal transplant studies in the 1990s. NeuroGeneration Inc.'s Phase II trial of
its neural stem cell-derived dopaminergic cell therapy has been on hold since
2008 due to cell manufacturing concerns. Last month, Geron Corp. discontinued its ESC therapy program, which
included a Phase I trial of oligodendrocyte progenitor cells in acute spinal
cord injury (SCI).

Two
companies-BrainStorm Cell Therapeutics Inc. and International Stem Cell Corp.-have preclinical
programs to generate stem cell-derived therapies for PD. Those approaches use mesenchymal
and parthenogenetically derived stem cells, respectively, but these cell types
are not thought to be as readily programmable into specific neurons as ESCs.

Isacson,
Freed and Studer all noted that the complexity of the regulatory path and
pessimism about stem cell therapies mean that, in the short term, the likeliest
commercial use for the new dopaminergic cells would be in in vitro drug
screening assays.

"Because
these cells are much closer to the real dopaminergic cells [than previous
cells], they are suitable for drug screening," said Studer.

Cellular reagent company Cellular Dynamics International Inc.
is negotiating a license to Studer's technology. Chris Parker, VP and chief
commercial officer of CDI, noted that Studer's cells would fit well with the
company's portfolio of specialized neuronal cells for drug screening.

Parker
said that to be commercially useful, Studer's protocol would need to be scaled
up at least 1,000-fold, but such scaling often requires major changes to the
cell culture protocols.

"In
this paper, they make millions of cells per animal, but we would have to make
billions and billions of cells for this to be useful as a screening platform,"
said Parker.

Scaling
up the procedure will require considerable refinement.

"If
you're going to make a product, you have to know how many of these cells will
survive freezing and thawing, how many will stick to a matrix, how many of them
form the appropriate cell type," said Parker. "For every cell line,
the protocol must be optimized and modified."

Studer
said his next step is to scale up production of his dopaminergic neurons.

He
has filed a patent on his methods, which is available for licensing.

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